The Adeno-associated viruses (AAVs) are not associated with any diseases and their ability to package non-genomic DNA and to transduce different cell/tissue populations for corrective gene delivery has generated significant interest in understanding their basic biology. This includes their capsid structure, cellular tropism and interactions for entry, uncoating, replication, DNA packaging, capsid assembly, and antibody neutralization. The goal is to improve their specificity and efficacy as vectors. However, while the majority of the characterization of the AAVs has been directed at serotype 2 (AAV2), studies on some of the more recently identified antigenically distinct human and primate viruses show enhanced transduction properties for particular cell types compared to AAV2. This property is mediated by their capsid sequence. Thus while providing the gene therapy community with a more expansive choice of potential AAV vectors for development, specific tissue/organ targeting for improving safety profiles and efficacy as well as engineering a faster onset of transduction would be greatly aided by identifying the capsid features of the other AAVs that correlates with their distinct tissue tropism and transduction phenotypes as well as their antigenic reactivities. The overall objective of this proposal is a structure-function analysis of the AAV capsid to identify features that (I) determine differential cell tropism;(II) affect transduction efficiency, (III) and are utilized for cell receptor recognition. The analysis will also provide information on capsid features that are conserved and thus could be important for the fidelity of viral capsid assembly interactions, and on capsid regions that dictate the distinct antigenicity of the AAV clade groups. Genetically manipulating these features could give rise to a new generation of corrective viral gene delivery vectors with synergistic improvements in tissue tropism and specificity, transduction efficiencies, and the ability to evade existing host immune responses. To achieve our objectives, we will determine the capsid structures for representative members of the AAV clade groups, alone and in complex with identified carbohydrate receptors, by X-ray crystallography, and functionally annotate AAV capsid regions involved in the receptor interactions using mutagenesis, cell binding and transduction assays, and biophysical measurements of binding affinity. We have developed a scaleable baculovirus system for the expression of wild type and mutant virus capsids for these studies.